The invention relates to the separation of transmissible spongiform encephalopathies (TSEs), or prions, from biological materials. The prions are separated from biological materials by precipitation with a polyalkylene glycol, such as polyethylene glycol.
Transmissible spongiform encephalopathies (TSEs), or pathogenic prions, cause fatal neurodegenerative diseases in humans and other mammalian species, particularly sheep, goats, cattle, mink, elk, and deer. The human forms of the disease include Creutzfeldt-Jakob disease (CJD), Gerstmann-Strxc3xa4ussler-Scheinker (GSS) syndrome, fatal familial insomnia (FFI), and Kuru. Symptoms of these diseases include myoclonus, ataxia, and progressive dementia. The pathology of these diseases includes the formation of amyloid plaques in the brain.
The occurrence of prion diseases has been linked to infectious transmission, as well as genetic and sporadic, causes. For example, Kuru, which afflicted the Fore tribe of New Guinea, was caused by the ingestion of infected brain tissue of other tribal members during ritualistic cannibalism. Alpers (1979) Slow Transmissible Diseases of the Nervous System, Vol. 1, S. B. Pruisner and W. J. Hadlow, eds. (New York: Academic Press), pp. 66-90. More recently, the occurrence of variant CJD in the United Kingdom has been postulated to be the result of consumption of beef from cattle infected with a TSE. G. Chazot, et al., (1996) Lancet 347:1181; R. G. Will, et al., (1996) Lancet 347:921-25. Similarly, iatrogenic CJD has been caused by the injection of human growth hormone derived from cadaveric pituitaries. Brown, et al., (1992) Lancet 340:24-27. Instances of infectious activity of prions, as noted above, suggest that prion infectivity may be transmitted through biological materials.
Prions are believed to infect humans and other mammals via transmission of a protein, the prion protein, without concomitant transmission of a nucleic acid. It has been postulated that the infectious agent is a disease-causing variant of the naturally-occurring, cellular prion protein (PrPC), the physiologic function of which is presently not known. In this theory, the variant form of the prion protein (PrPSc) induces structural changes in the normal form of the prion protein (PrPC), thereby converting it to the disease-causing form of the protein (PrPSc).
Therapeutic compositions are often derived from biological materials. For example, therapeutically valuable proteins such as Factor VIII, xcex1-l proteinase inhibitor, and albumin are isolated from human plasma. Other infectious agents, such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV) have been known to be transmitted by therapeutic use of blood or blood products that have not undergone inactivation methods or procedures that effectively remove the agents from the product processing stream. Evidence of the transmission of TSEs through other biological materials raises concern over possible transmission of TSEs through blood and blood-derived products, recombinant products, and other products of biological orgin.
Known methods for reducing the risk of infection from blood borne pathogens, such as HIV and HCV, however, are not effective at inactivating prions. Furthermore, methods that do reduce the risk of infection from prions, which include treatment with strong base and/or autoclaving, are not suited for use with procedures for the preparation of therapeutic proteins, because such procedures denature the proteins.
A method for separating prions from biological materials, such as blood plasma fractions and aqueous solutions of recombinantly produced proteins, is therefore needed. The present invention provides a method of separating prions from such biological materials without denaturing therapeutically valuable proteins.
The invention provides methods for the separation of prions from biological materials. Biological materials include biologically derived fluids, such as blood plasma fractions, biological samples, such as samples of tissues, and aqueous solutions of recombinantly produced products, such as recombinant proteins. Generally, the methods provide for the separation of prions from the biological material by adding a polyalkylene glycol to the biological material to obtain a mixture of the polyalkylene glycol and the biological material. From this mixture a precipitate containing the prion is formed. This precipitate is separated from the mixture, such as by filtration or centrifugation, to obtain a purified biological material.
The polyalkylene glycol for use in methods of the invention may be polyethylene glycol (PEG) or polypropylene glycol (PPG), but PEG is preferred. The PEG is added to the biological material so that the weight of PEG per volume of the resulting mixture is at least about 2%. The weight of PEG per volume of resulting mixture is preferably about 3% to about 15%, and is more preferably greater than or equal to about 10%. The PEG for use in methods of the invention has a molecular weight of between about 2,000 and about 10,000, and preferably has a molecular weight of about 3,350.
The method of the invention may be used to separate prions from blood plasma fractions. When used to separate prions from blood plasma fractions. the method of the invention results in at least 3 logs of clearance of prions from the blood plasma fraction.
The method of the invention may be used to separate prions from aqueous solutions of recombinantly produced products. A recombinantly produced product may be a recombinantly produced protein.
In preferred methods of the invention, a prion is removed from a blood plasma fraction including the prion by adding PEG to the blood plasma fraction so that the mixture obtained is at least about 10% weight per volume PEG. From this mixture, the prion is precipitated. The precipitate, including the prion, is then separated from the mixture. In this embodiment, the PEG preferably has a molecular weight of about 3,350 and is preferably 11.5% weight per volume of the resulting mixture.
The present invention therefore provides methods for the separation of prions from biological materials, such as blood plasma fractions, without using harsh conditions that often denature therapeutically valuable proteins. Such methods reduce the risk of transmission of these infectious prions from biological materials, which are then further processed for therapeutic use. These and other aspects of the invention will be more apparent from the following description and claims.
Methods of the invention provide for the separation of prions from biological materials, such as biologically derived fluids, biological samples, blood plasma fractions. and solutions of recombinantly produced products, such as proteins. Methods of the invention provide for the precipitation of prions from the biological material by adding a polyalkylene glycol, such as polyethylene glycol (PEG), to the biological material. The precipitate containing the prions is then separated from the biological material, such as by filtration or centrifugation, for example.
PEG for use in methods of the invention has a molecular weight of between about 2,000 and about 10,000, and preferably has a molecular weight of about 3,350. The PEG is added to the biological material so that the weight of PEG per volume of the resulting mixture is at least about 2%. The PEG is preferably between about 3% and about 15% of the resulting mixture, and is more preferably greater than or equal to about 10%.
As described above, the biological material for use in methods of the invention may be a biologically derived fluid, a biological sample, a blood plasma fraction, or a solution of a recombinantly produced product. A biologically derived fluid includes, but is not limited to, solutions derived from biological materials, and body fluids, such as cerebrospinal fluid, lymphatic fluid, plasma, etc. A biological sample includes, but is not limited to, body tissues and solutions of body tissues, such as central nervous system tissue (e.g., brain), spleen, tonsils, etc. Such solutions preferably do not contain a detergent. Blood plasma fractions include any portion or all of plasma derived from the blood. Finally, a solution of a recombinantly produced product includes, but is not limited to, aqueous solutions of recombinantly produced proteins or proteins produced by transgenic animals.
The prion protein (PrP) is present in its normal, cellular form (PrPC) in most biological materials, and is particularly prevalent in central nervous system (CNS) tissue. The physiological function of PrPC is, however, not known. In theory, PrPC undergoes a conformational change in the diseased state. Evidence for this theory is the presence of amyloid plaques containing an abnormally folded form of PrP in the brains of TSE victims. This conformational change alters the physicochemical properties of the protein. thereby rendering it less soluble in physiological fluids and more proteinase resistant. This altered form of the protein is typically designated PrPSc (prion protein scrapie) and is believed to be the infectious agent, or, at least, necessary for infection. Removal of PrPSc from biological materials may,, therefore, decrease the risk of transmission of TSEs.
A correlation between use of infected biological samples or biologically derived fluids and the transmission of prion disease has been demonstrated. For example, prion diseases have been transmitted through therapeutic use of infected body tissues, such as corneas, and biologically derived fluids, such as solutions of human growth hormone derived from cadaveric pituitaries. These concerns over the risk of transmission of prion disease through the use of biological materials can be addressed by the methods of the invention, as shown in the examples below.